Sample sizes
The sample sizes were not statistically determined before the experiments. Rather, the group sizes were on the basis of published literature for the type of manipulation (chemogenetics, site-specific genetic knockout and pharmacology) and measured outcome (such as pain behaviours) published in the field and/or by the authors involved2,28,64,66. The sample sizes for all experiments are included in the figure legends.
Data exclusions
For all imaging and behavioural studies, virus-injected animals with either little or no evidence of viral transduction and/or incorrect viral targeting were excluded from any final analyses. No other mice or data points were excluded across analyses.
Replication
For many of the behavioural studies, several cohorts were used owing to the large number of animals in the final group sizes. All behaviour results were consistent and replicated across cohorts. Individual data points or lines were included, indicating consistent trends across many mice in each behavioural study.
Blinding
Mice were randomly assigned into control or experimental groups to the best of the experimenter’s abilities, with counterbalancing for age and sex as needed. In most of the included studies, the experimental and control groups differed only in the type of virus infused intracranially. The surgical protocol for all mice was identical in the amount, wait time and location of the intracranial injection. Each surgery day was randomly assigned as a control or experimental surgery date, and the corresponding mice from the predetermined groups underwent surgery on that day. GRIN lens and fibre placements and viral spread maps were included in the supplement to demonstrate the similarity of the injection protocol and outcome. Once the experimental and control groups were formed to comprise the study cohort of mice, the cohort underwent all behavioural testing concurrently, and experimenters were blinded. After the analyses were completed, the experimenters were unblinded.
Animals
All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania and performed in accordance with the US National Institutes of Health guidelines. Male and female mice aged 2–5 months were housed two to five per cage and maintained on a 12-h reverse light–dark cycle in a temperature-controlled and humidity-controlled environment. All experiments were performed during the dark cycle. The mice had ad libitum food and water access throughout the experiments. For behavioural, anatomical and transcriptomic experiments, we used Fos–FOS–2A–iCreERT2 or ‘TRAP2’ mice (Fostm2.1(icre/ERT2)Luo)Luo; The Jackson Laboratory; stock no. 030323)69 bred to homozygosity, C57BL/6J mice (The Jackson Laboratory; stock no. 000664), Oprm1Cre/Cre mice (B6.Cg–Oprm1tm1.1(cre/GFP)Rpa/J; The Jackson Laboratory; stock no. 035574) and Oprm1fl/fl mice (B6;129–Oprm1tm1.1Cgrf/KffJ; The Jackson Laboratory; stock no. 030074). Further anatomical experiments used TRAP2 mice crossed with Ai9 (B6.Cg–Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J; The Jackson Laboratory; stock no. 007909) reporter mice that express a tdTomato fluorophore in a Cre-dependent manner.
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